The clearly defined genetic and phenotypic stages in the evolution of squamous tumor development on mouse skin provides an opportunity to evaluate mechanisms of carcinogenesis in a stage-specific manner. This project is designed to identify genetic loci that modify the later stages of cancer development and elucidate the pathways through which they function. We have focused on two major pathways that are relevant to human cancer, the p53 and AP-1 pathways that regulate gene expression. Upregulation of c-Fos is detected in squamous tumors that are progressing from benign to malignant, and deletion of c-Fos prevents malignant conversion. We have targeted a dominant-negative mutant Fos protein to the epidermis of transgenic mice using a tetracycline conditional promoter system. Expression of the mutant in utero is embryonic lethal while expression in the adult produces a skin phenotype. A series of clonal c-Fos null keratinocyte cell lines have been characterized and fail to produce tumors in vivo when transformed by a ras oncogene. By selecting for genes that are regulated by p53 in keratinocytes, we have cloned mtCLIC, a chloride channel protein of mitochondria. mtCLIC causes apoptosis in normal and neoplastic keratinocytes when overexpressed, and antisense mtCLIC prevents apoptosis induced by overexpressing p53. mtCLIC also increases after DNA damage in keratinocytes and synergizes with Bax to induce apoptosis. During tumor progression, the expression of mtCLIC decreases in dysplastic and malignant epidermal tumors, and this is associated with mutations in p53. The human mtCLIC gene is located on chromosome 1p32-35, a site that is frequently deleted or translocated in human epithelial malignancies.